WO2014126332A1 - Domaine de transduction protéique fondé sur un conjugué d'aptamères-nanoparticules d'or et procédé de production correspondant - Google Patents

Domaine de transduction protéique fondé sur un conjugué d'aptamères-nanoparticules d'or et procédé de production correspondant Download PDF

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WO2014126332A1
WO2014126332A1 PCT/KR2013/011851 KR2013011851W WO2014126332A1 WO 2014126332 A1 WO2014126332 A1 WO 2014126332A1 KR 2013011851 W KR2013011851 W KR 2013011851W WO 2014126332 A1 WO2014126332 A1 WO 2014126332A1
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protein
aunp
apt
bim
results
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Korean (ko)
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이강석
배지현
성맹제
한민수
염지현
유상미
하남출
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중앙대학교 산학협력단
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Priority to EP13875242.3A priority Critical patent/EP2995300B1/fr
Publication of WO2014126332A1 publication Critical patent/WO2014126332A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers

Definitions

  • the present invention relates to a protein carrier and a method for preparing the same, and more particularly, to a protein carrier having an improved protein delivery capacity into cells by binding aptamers or protein specific aptamers of various tags to gold nanoparticles, and a method for preparing the same. .
  • nanoparticles have been used as promising tools in biomedical fields such as drug delivery, gene delivery, intracellular imaging, and phototherapy, in particular gold nanomaterials are easy to synthesize, act on, chemical stability and biocompatibility. Attention has been paid to due to the fact that the optical and electrical characteristics are adjustable.
  • materials prepared for diagnosis or treatment have to be delivered into the cell in a diagnostic or therapeutically effective amount thereof, and since the cells are impermeable to large molecules such as proteins, there is a limit to diagnosis or treatment using such proteins. There is a problem.
  • An object of the present invention is to provide a protein carrier comprising an aptamer coupled to the surface of the gold nanoparticles and the gold nanoparticles in order to solve the problems of the prior art as described above.
  • the present invention is to provide a method for producing a protein carrier comprising the step of binding the gold nanoparticles and aptamer (aptamer).
  • the present invention provides a protein carrier comprising gold nanoparticles and an aptamer bound to the surface of the gold nanoparticles.
  • the gold nanoparticles are characterized in that having a size of 10-20 nm.
  • the aptamer is characterized in that it specifically binds to the protein to be delivered.
  • the protein is characterized in that the protein labeled with a tag (tag).
  • the aptamer is characterized in that it specifically binds to the label.
  • the label is characterized in that the histidine and / or glutathione S-transferase (GST; glutathione S-transferase).
  • GST glutathione S-transferase
  • the present invention provides a method for producing a protein carrier comprising the step of binding the gold nanoparticles and aptamers.
  • the present invention provides a complex consisting of a protein carrier and a protein.
  • the present invention provides a protein delivery method comprising the step of administering the complex to a subject.
  • the protein carrier according to the present invention is capable of effectively delivering proteins into cells by specifically binding to proteins to be delivered by aptamers or protein-specific aptamers of various tags bound to gold nanoparticles, and having very low cytotoxicity. Not only harmless to the human body by using the particles, but also reflects light at various wavelengths can easily determine the location in the cell, it is expected to be useful in the diagnosis or treatment of diseases.
  • FIG. 1 is a schematic diagram illustrating the production of aptamer-gold nanoparticle-protein complexes by binding His-aptamer and His-tagged protein to metal nanoparticles.
  • Figure 2 shows the results of confocal microscopy analysis of E. coli AcrA protein delivery labeled secondary antibody Rabbit-488 (Green).
  • Figure 3 shows the results of confocal microscopy analysis of E. coli AcrA protein delivery labeled secondary antibody Rabbit-584 (Red).
  • Figure 4 shows the results of confocal microscopy analysis of AcrA protein delivery when HeLa cells were treated with AcrA or His-AcrA protein with or without binding AuNP-His-Apt.
  • Figure 5 shows the results of Western blotting analysis for detecting E. coli AcrA protein.
  • Figure 6 shows the results of Western blotting analysis for AcrA protein delivery when HeLa cells were treated with AcrA or His-AcrA protein with or without AuNP-His-Apt binding.
  • Figure 7 shows the results of confocal microscopy analysis of BCL-xL protein delivery labeled secondary antibody Rabbit-488 (Green).
  • Figure 10 shows the results of Western blotting analysis for BCL-xL protein delivery when BCL-xL or His-BCL-xL protein with or without binding AuNP-His-Apt to HeLa cells.
  • FIG. 11 shows flow cytometry results for BCL-xL protein delivery labeled with Alexa488.
  • FIG. 12 shows flow cytometry results for the delivery of cy5 (Red) labeled His-aptamer gold nanocarriers and Alexa488 (Green) labeled BCL-xL proteins.
  • FIG. 13 shows flow cytometry results of BCL-xL protein delivery when HeLa cells were treated with BCL-xL or His-BCL-xL proteins with or without AuNP-His-Apt binding.
  • Figure 14 shows the results of confocal microscopy analysis of FOXL2 protein delivery labeled with secondary antibody Rabbit-Rodamine (Red).
  • FIG. 16 is a diagram showing the results of analysis of confocal microscopy of HeLa cells treated with AuNP-GST-Apt-GST-FOXL2 to confirm internalization of GST-FOXL2 protein by AuNP-GST-Apt. .
  • Figure 17 shows the results of Western blotting analysis of HeLa cells treated with AuNP-GST-Apt-GST-FOXL2 and cultured to confirm internalization of GST-FOXL2 protein by AuNP-GST-Apt.
  • FIG. 18 is a diagram showing the result of Western blotting of nuclear extract in order to confirm the nuclear localization of GST-FOXL2.
  • FIG. 19 shows the results of real time-PCR using cDNA library synthesized from RNA isolated from HeLa cells cultured with AuNP-GST-Apt-GST-FOXL2.
  • 20 is a view showing the results of confirming the transmission of BIM protein in HeLa cells by confocal microscopy by the gold nanoparticle-aptamer conjugate (AuNP-His-Apt) of the present invention.
  • 21 is a diagram showing the results confirmed by flow cytometry the delivery of BIM protein in HeLa cells by the gold nanoparticle-aptamer conjugate (AuNP-His-Apt) of the present invention.
  • FIG. 22 is a diagram showing the results confirmed by Western blotting analysis of the delivery of BIM protein in HeLa cells by the gold nanoparticle-aptamer conjugate (AuNP-His-Apt) of the present invention.
  • Figure 23 is a diagram showing the results confirmed by flow cytometry the delivery of BIM protein in HeLa cells by the gold nanoparticle-aptamer conjugate (AuNP-His-Apt) of the present invention.
  • 24 is a diagram showing the results of confirming cell viability after treatment with HeNP cells in AuNP-His-Apt-His-BIM.
  • 25 is a view showing the results of confirming the transmission of BIM protein in human primary granulosa cells by the gold nanoparticle-aptamer conjugate (AuNP-His-Apt) of the present invention by confocal microscopy.
  • FIG. 26 is a diagram illustrating the results of Western blotting analysis of BIM protein delivery in human primary granulosa cells by the gold nanoparticle-aptamer conjugate (AuNP-His-Apt) of the present invention.
  • FIG. 26 is a diagram illustrating the results of Western blotting analysis of BIM protein delivery in human primary granulosa cells by the gold nanoparticle-aptamer conjugate (AuNP-His-Apt) of the present invention.
  • FIG. 27 is a diagram showing the results of confirming the transmission of BIM protein in KGN cells by confocal microscopy by the gold nanoparticle-aptamer conjugate (AuNP-His-Apt) of the present invention.
  • FIG. 28 is a diagram showing the results confirmed by Western blotting analysis of the delivery of BIM protein in KGN cells by the gold nanoparticle-aptamer conjugate (AuNP-His-Apt) of the present invention.
  • ESA endosomal marker protein
  • LAMP1 lysosomal-associated membrane protein 1
  • FIG. 31 shows the results of flow cytometry analysis of the cells of FIG. 29 in the presence or absence of FBS.
  • FIG. 32 is a diagram showing the results obtained by treating AuNP-His-Apt-His-BIM to HeLa cells and confirming their cross section through Transmission Electron Microscopy (TEM).
  • TEM Transmission Electron Microscopy
  • FIG. 33 shows the results of quantitative analysis of aptamers to evaluate the aptamer loading capacity of gold nanoparticles (AuNP).
  • Fig. 34 shows the results of measuring protein binding ability of AuNP-His-Apt or AuNP-GST-Apt.
  • 35 is a diagram showing the results of measuring cell viability after treatment with HeLa cells at different AuNP-His-Apt concentrations (0, 0.5, 1.0 and 2 nM) and cultured.
  • FIG. 37 shows the results of measurement of tumor volume and weight change over time by AuNP-His-Apt-His-BIM complex injection in an in vivo mouse xenograft model.
  • FIG. 38 shows confocal microscopy of His-BIM delivery to tumor tissues by AuNP-His-Apt conjugates in an in vivo mouse xenograft model.
  • 40 shows the results confirmed by immunohistochemical analysis of his-BIM delivery to tumor tissues by AuNP-His-Apt conjugates in an in vivo mouse xenograft model.
  • FIG. 41 shows the results of confirming the target delivery ability of proteins by AuNP-His-Apt in an in vivo mouse xenograft model by confocal microscopy.
  • FIG. 42 shows the results of measuring the relative intensity of fluorescence in tumor and organ cross sections using Image J software to confirm target delivery ability of AuNP-His-Apt protein in an in vivo mouse xenograft model. .
  • FIG. 43 shows the results of immunohistochemical analysis of tumor tissues using anti-BIM antibodies to confirm target delivery ability of proteins by AuNP-His-Apt in an in vivo mouse xenograft model.
  • FIG. 45 shows the results of measuring the relative intensities of fluorescence in tissues using Image J software to confirm biodistribution of the AuNP-His-Apt-Alexa488-His-BIM complex.
  • Figure 46 shows the results of confirming the change in the release of AST and LDH content by AuNP-His-Apt-Alexa488-His-BIM to confirm the biodistribution of AuNP-His-Apt-Alexa488-His-BIM complex The figure shown.
  • the present inventors have completed the present invention as a result of studying a protein carrier having very little cytotoxicity and excellent protein delivery ability into cells.
  • the present invention provides a protein carrier comprising gold nanoparticles and an aptamer bound to the surface of the gold nanoparticles.
  • nanoparticles means particles of various materials having a diameter in nano units, and the nanoparticles are not particularly limited as long as the particles have nano-size particles.
  • gold nanoparticle refers to a metal particle of gold having a diameter in nano units, and such small particle size facilitates the penetration of nanoparticles of the present invention into cells of interest (eg, human cells). Thereby allowing the penetration of protein carriers into the cell.
  • Gold nanoparticles are not only easy to manufacture in the form of stable particles, but also can be varied in size from 0.8 nm to 200 nm.
  • gold can be combined with various kinds of molecules such as peptides, proteins, nucleic acids, etc., to modify its structure, and reflect light at various wavelengths.
  • gold nanoparticles have advantages in that they are harmless to the human body and have high biocompatibility, and have very little cytotoxicity, unlike heavy metals such as manganese, aluminum, cadmium, lead, mercury, cobalt, nickel, and beryllium.
  • the gold nanoparticles When the gold nanoparticles are larger than 100 nm in diameter, their properties as nanoparticles disappear, and the bonding of functional groups such as thiol groups and the surface of gold without nanomaterials is weakened, and the diameter is other than 10-20 nm. Since gold nanoparticles cause cytotoxicity, the gold nanoparticles of the present invention preferably have a diameter of 10 to 20 nm.
  • aptamer means a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) which has a stable tertiary structure and has a characteristic that can bind to a target molecule with high affinity and specificity.
  • SELEX Systematic Evolution of Ligands of Exponential enrichment
  • the aptamer binding to the gold nanoparticles may use any kind of aptamer or protein specific aptamer of various tags, and is not particularly limited.
  • the protein carrier according to the present invention can effectively deliver proteins into cells by specifically binding to proteins to be delivered by aptamers or protein-specific aptamers of various tags bound to gold nanoparticles, which is useful for diagnosing or treating diseases. Can be used.
  • the present invention provides a complex made by combining the protein carrier and the protein.
  • the present invention also provides a protein delivery method comprising administering the complex to a subject.
  • subject refers to a subject in need of diagnosis or treatment of a disease, and more particularly human or non-human primates, mice, rats, dogs, cats, horses and cattle Means such mammals.
  • the histidine-tag aptamer or GST-tag aptamer to gold nanoparticles to prepare a protein carrier (see Example 2), AcrA protein by the protein carrier (Example 3)
  • the cell delivery capacity of the BCL-xL protein (Example 4), FOXL2 protein (Example 5), and BIM protein (Example 7) was confirmed, and the protein delivery system of the present invention confirmed that the proteins were effectively delivered into cells. It was.
  • the protein carrier (gold nanoparticle-aptamer conjugate) of the present invention is not only cytotoxic (see Example 10), but also can simultaneously deliver a plurality of proteins ( See Example 11).
  • antitumor activity see Example 12
  • in vivo distribution see Example 14
  • His-BIM protein was effectively delivered by AuNP-His-Apt-His-BIM injection in vivo .
  • HeLa Human cervical carcinoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM).
  • Human adult-type granulosa cell tumor-derived KGN and primary rat granulosa cells were cultured in Dulbecco's modified Eagle's medium / F12. All media contain 10% heat-inactivated fetal bovine serum (Caisson, USA) and 1% penicillin-streptomycin (Welgene, Korea).
  • the gold nanoparticle-aptamer conjugate was pre-incubated at 80 ° C. for 5 minutes to prevent secondary structure formation.
  • AuNP-His / GST-Apt (1 nM) and purified His / GST-tagged protein were reacted for 10 minutes at room temperature in 1 ⁇ PBS containing 5 mM MgCl 2 (pH7.2) and then centrifuged at 13,000 ⁇ g. The supernatant was removed after separation.
  • AuNP-Apt-protein complexes were washed using TBST with 500 mM NaCl and 1M KCl.
  • HeLa cells (1 x 10 7 ) 18-20 g of 6-week-old immunodeficiency BALB / c-nu / nu mice (Central Lab Animal Inc, Korea) were injected subcutaneously and the AuNP-GST-Apt-His-BIM or AuNP-His-Apt-His-BIM complex ( complex) was injected directly into tumors (tumor volume ⁇ 0.1 cm 3 ). The length of the long and short axis of the tumor was measured daily. Mice were sacrificed 30 days after the initial infusion of the complex and tumor samples were collected for further analysis. Results were expressed as mean ⁇ SEM of tumors from 5 mice in each group, with * marks indicating significant values when compared to the corresponding control (P ⁇ 0.05).
  • Histidine-tag added protein was generated in BL21 (DE3) E. coli cells and purified by Ni-NTA agarose resin (Qiagen, USA) or GST Sepharose 4B bead (GE Life science, USA). To remove His-tag or GST-tag from the purified protein, TAGZyme system (Qiagen) and Thrombin (GE Life science) were used. For protein uptake experiments, proteins were labeled with Alexa Fluor® 488 Microscale Protein Labeling Kit (Invitrogen, USA) or Texas Red® X Protein Labeling Kit (Invitrogen).
  • HeLa cells were seeded in 96-well plates and incubated overnight to allow the cells to adhere. Cells were incubated with AuNP-His-Apt-His-BIM and cell viability was measured (Yeom, JH et al. Inhibition of Xenograft Tumor Growth by Gold Nanoparticle-DNA Oligonucleotide Conjugates-Assisted Delivery of BAX mRNA.PLoS One 8, e75369 (2013)).
  • HeLa cells were treated with AuNP-His-Apt-His-BIM and cultured for 1 hour, and then mitochondrial fractions were obtained using a mitochondria isolation kit (Thermo, USA).
  • HeLa cells were treated with AuNP-GST-Apt-GST-FOXL2 for 1 hour, and then nuclear fractions were separated using NE-PER® nuclear and cytoplasmic extraction reagents (Thermo).
  • Reverse transcription and real-time PCR were performed according to known methods (Kim, JH et al. Differential apoptotic activities of wild-type FOXL2 and the adult-type granulosa cell tumor-associated mutant FOXL2 (C134W). Oncogene 30, 1653-1663 (2010)).
  • Protein carriers were prepared using his-tag aptamer and glutathione S-transferase (GST) aptamers, the schematic diagram of which is shown in FIG. 1.
  • a histidine that detects and specifically binds thereto was used. More specifically, the histidine-tag DNA aptamer is 3 'A terminal is a thiol group modified aptamer, consisting of the nucleotide sequence of 5'-GCTATGGGTGGTCTGGTTGGGATTGGCCCCGGGAGCTGGCAAAAAAAA-3' (SH) (SEQ ID NO: 1.)
  • SH thiol group modified aptamer
  • DNA aptamer pretreated and precipitated in Example 2-1 a was dissolved in water, and then added to gold nanoparticles, and the present inventors used gold nanoparticles (AuNP) as gold colloids purchased from BBI Life Science, UK. -15 nm (# EM.GC15) was used. Meanwhile, to confirm localization of gold nanoparticles (AuNP), gold nanoparticles (AuNP) were combined with 5'-Cy5-labeled His-aptamer (Bioneer).
  • a glutathione S-transferase (GST) was detected and a DNA aptamer (GST-tag aptamer) that specifically binds thereto was used.
  • GST-tag DNA aptamer is an aptamer modified with a thiol group at the 3 'end, and is an aptamer composed of a nucleotide sequence of 5'-CTGCCCCGCTATAGAACACCCGTTGGGCAAATGTGTTCGAAAAAAAAA-3' (SH) (SEQ ID NO: 2).
  • the GST-tag DNA aptamer was pretreated in the same manner as 2-1 a).
  • DNA aptamer pretreated and precipitated through a) of Example 2-2 was combined with gold nanoparticles in the same manner as b) of Example 2-1 to obtain a final aptamer-gold conjugate.
  • the gold nanoparticle-aptamer conjugate 1 nM prepared in Example 2-1 was treated at 80 ° C. for 5 minutes, and then cooled to room temperature. 1-2 ⁇ g / ⁇ l histidine-tag-added AcrA (His-AcrA) or histidine-tag-free AcrA protein (AcrA) was added to 5 mM MgCl 2 -PBS buffer and reacted at room temperature for 2 hours. . The reacted gold nanoparticle-aptamer-protein complex was centrifuged at ⁇ 10,000 ⁇ g for 20 minutes to remove supernatant.
  • the gold nanoparticle-aptamer-protein complex from which the supernatant was removed was treated with a human uterine cancer cell line (HeLa cell) and then incubated for 1 hour. After incubation, washed with PBS, fixed with 4% paraformaldehyde (paraformaldehyde), and the immobilized samples were immunostained (observe) with a confocal microscope (confocal microscope), the results are shown in Figures 2 and 3 Indicated.
  • Figure 2 shows the results of confocal microscopy analysis of Escherichia coli AcrA protein delivery using a secondary antibody labeled Rabbit-488 (Green)
  • Figure 3 is a secondary antibody labeled Rabbit-584 (Red)
  • Confocal microscopic analysis of E. coli AcrA protein delivery is shown. Since the histidine-tag aptamer in histidine-tag aptamer-gold nanoparticle conjugate can detect and specifically bind histidine, the histidine-tag aptamer-gold nanoparticle conjugate is shown in FIGS. 2 and 3. When combined with the histidine-tag added AcrA protein was confirmed that the intracellular protein transfer ability is very excellent than when combined with the histidine-tag removed AcrA protein.
  • AcrA or His-AcrA protein with or without binding AuNP-His-Apt was treated in HeLa cells and incubated for 1 hour, followed by Western blotting analysis using anti-AcrA antibody. 6 is shown.
  • Figure 7 shows the results of confocal microscopy analysis of BCL-xL protein delivery labeled secondary antibody Rabbit-488 (Green), as shown in Figure 7, histidine-tag aptamer-gold nano The binding of the particle conjugate to the BCL-xL protein to which the histidine-tag was added was confirmed to be superior to the intracellular protein delivery ability compared to the BCL-xL protein to which the histidine-tag was removed.
  • BCL-xL or His-BCL-xL proteins with or without binding AuNP-His-Apt were treated in HeLa cells and incubated for 1 hour, followed by mouse IgG labeled with anti-BCL-xL and Alexa 488. Immunostaining was carried out using the same, and confirmed by confocal fluorescence microscopy, and the results are shown in FIG. 8. Representative confocal fluorescence images were obtained from a 40 ⁇ water immersion objective, with a scale bar of 20 ⁇ m.
  • Figure 9 shows the results of Western blotting analysis for the detection of BCL-xL protein, as shown in Figure 9, histidine-tag aptamer-gold nanoparticle conjugate BCL-xL with histidine-tag added When combined with the protein (lane 3), it was confirmed that the BCL-xL protein is detected.
  • BCL-xL or His-BCL-xL proteins with or without AuNP-His-Apt were treated in HeLa cells and incubated for 1 hour, followed by Western blotting analysis. Indicated.
  • Composites were prepared in the same manner as -1. After preparation of the complex, the aptamer-gold nanoparticle-protein complex from which the supernatant was removed was treated with a human uterine cancer cell line (HeLa cell) and then incubated for 1 hour. After incubation, the cells were washed with PBS and dispersed again in 1 ml PBS buffer. Samples were observed using flow cytometry and the results are shown in FIGS. 11 and 12.
  • Figure 11 shows the flow cytometry results for the delivery of His-BCL-xL protein and BCL-xL protein labeled with Alexa488 (Green) fluorescent material
  • Figure 12 is a gold nano-labeled cy5 (Red)- Flow cytometer showing the number of cells having both the red wavelength of the gold nanoparticles and the green wavelength of the protein by constructing a His-BCL-xL protein and a BCL-xL protein complex labeled with an Alexa488 (Green) phosphor on the aptamer conjugate The results are shown.
  • BCL-xL or His-BCL-xL proteins with or without AuNP-His-Apt were treated with HeLa cells and incubated for 1 hour, followed by flow cytometry analysis of Alexa 488-positive cells. Is shown in FIG. 13.
  • Example 3-1 Intracellular delivery ability of GST-FOXL2 protein was confirmed in the same manner as in Example 3-1 with the gold nanoparticle-aptamer conjugate prepared in Example 2-2, and the results are shown in FIG. 14.
  • FIG. 14 shows the results of confocal microscopy analysis of FOXL2 protein delivery in which the secondary antibody is labeled with Rabbit-Rodamine (Red), and as shown in FIG. 14, the GST-tag aptamer-gold nanoparticle conjugate.
  • Red Rabbit-Rodamine
  • Example 2-2 Western blotting was performed in the same manner as in Example 3-2 with the gold nanoparticle-aptamer conjugate prepared in Example 2-2 to confirm the intracellular delivery capacity of the GST-FOXL2 protein. It is shown in 15.
  • FIG. 15 shows the results of Western blotting analysis for FOXL2 protein detection.
  • the GST-tag aptamer-gold nanoparticle conjugate was combined with the FOXL2 protein to which the GST-tag was added. In some cases (lane 3), it was confirmed that FOXL2 protein was detected.
  • the aptamer-gold nanoparticle-protein complex (AuNP-GST-Apt-GST-FOXL2) prepared by reacting AuNP-GST-Apt and FOXL2 protein (GST-FOXL2) to which GST-tag was added was prepared, and this was HeLa. Cells were incubated for 1 hour and then immunostained using anti-FOXL2 and Alexa546-rabbit IgG. Cells were then observed under confocal fluorescence microscopy, and the results are shown in FIG. 16. At this time, the image was obtained from a 40 x water immersion objective, excitation at 556 nm and emission at 573 nm, with a scale bar of 20 ⁇ m.
  • FOXL2 was effectively delivered into HeLa cells. It was also confirmed that the FOXL2 protein was localized in the cell nucleus.
  • Real time-PCR was performed using a cDNA library synthesized from RNA isolated from HeLa cells treated with AuNP-GST-apt or AuNP-GST-apt-GST-FOXL2 and incubated for 1 hour. Shown in The results were expressed by normalizing by expression of glyceraldehydes 3-phosphate dehydrogenase (GAPDH) and folding the relative expression levels. The results (mean ⁇ SEM) were obtained from the results obtained by repeating three times. Statistically significant values are indicated as P ⁇ 0.05.
  • TNF-R1 tumor necrosis factor-receptor 1
  • Fas CD95 / APO-1
  • Example 7 Experiment of confirming intracellular delivery ability of BIM protein by gold nanoparticle-aptamer conjugate
  • Aptamer-gold nanoparticle-protein complex (AuNP-His-) prepared by adding histidine-tag to AuNP-His-Apt and AuNP-His-Apt and reacting BIM protein (His-BIM) labeled with Alexa 488 Apt-His-BIM) was treated in human uterine cancer cell lines (HeLa cells) and incubated for 1 hour, and then stained with Mitotracker (red) and DAPI, and observed by confocal fluorescence microscopy. The results are shown in FIG. It was.
  • the final concentrations of AuNP-His-Apt and His-BIM used were 1 nM and 0.4 ⁇ M, respectively, and the cells were visualized using an ax 40 water immersion objective, and the scale bar was 20 ⁇ m.
  • HeLa cells cultured with AuNP-His-Apt loaded with His-BIM protein (Alexa 488-His-BIM) labeled with Alexa 488 showed green fluorescence of Alexa 488, whereas AuNP It was confirmed that no signal was observed in cells treated with -His-Apt alone. In addition, it was confirmed that His-BIM protein delivered by AuNP-His-Apt was largely localized to mitochondria. From the above results, it was found that His-BIM protein was delivered into cells by the aptamer-gold nanoparticle conjugate of the present invention.
  • Alexa 488-His-BIM was treated with HeLa cells with or without reacting with AuNP-His-Apt, incubated for 1 hour, and observed using flow cytometry. The results are shown in FIG. 21. .
  • Aptamer-gold nanoparticle-protein complex (AuNP-His-Apt-His-BIM), which combines AuNP-His-Apt and His-BIM protein, was incubated in HeLa cells for 1 hour, and then Western blotting ) Analysis was performed and the results are shown in FIG. 22. Meanwhile, efficient separation of mitochondrial and cytoplasmic fractions was confirmed by Western blotting using anti-Cox IV and anti- ⁇ -actin, respectively.
  • His-BIM protein delivered by AuNP-His-Apt was delivered into cells, and particularly localized to mitochondria.
  • Annexin V-positive killer cells increased when AuNP-His-Apt-His-BIM was treated than the control group. From the above results, it was found that His-BIM protein was delivered into cells by the aptamer-gold nanoparticle conjugate of the present invention.
  • KGN cells were treated with AuNP-His-Apt (1 nM) or AuNP-His-Apt-Alexa 488-His-BIM complexes and incubated for 1 hour, and observed by confocal fluorescence microscopy. Indicated. At this time, the cells were visualized using an a ⁇ 40 water immersion objective, and the scale bar was 20 ⁇ m.
  • KGN cells were treated with AuNP-His-Apt (1 nM) or AuNP-His-Apt-Alexa 488-His-BIM complexes and incubated for 1 hour, followed by western blotting analysis. Is shown in FIG. 28.
  • aptamer-gold nanoparticle conjugate (Cy5-AuNP-His-Apt) labeled with Cy5 (Cyanine 5) fluorophore at the aptamer 5 'end and Cy5-AuNP-His-Apt and Alexa 488
  • the aptamer-gold nanoparticle-protein complex (Cy5-AuNP-His-Apt-His-BIM) prepared by reacting BIM protein (His-BIM) was treated with HeLa cells, incubated for 1 hour, and then confocal. Observation was made under a fluorescence microscope and the results are shown in FIG. 29. At this time, the cells were visualized using a 40 x water immersion objective, and the scale bar was 20 ⁇ m.
  • confocal fluorescence microscopic analysis of endosomal marker proteins (early endosome antigen 1 (EEA), or lysosomal-associated membrane protein 1 (LAMP1)) was performed, and the results are shown in FIG. 30. More specifically, AuNP-His-Apt-Alexa 488-His-BIM was treated with HeLa cells and incubated for 1 or 3 hours, and then treated with anti-EEA-1 (1: 500) and anti-LAMP1 (1: 100). The reaction was observed by confocal fluorescence microscopy. At this time, counterstain was performed with Alexa Fluor 546 goat anti-mouse IgG (1: 1,000) and Alexa Fluor 546 goat anti-rabbit IgG (1: 1,000).
  • EAA endosome antigen 1
  • LAMP1 lysosomal-associated membrane protein 1
  • the cells were subjected to flow cytometry in the presence or absence of FBS, and the results are shown in FIG. 31.
  • the percentages shown in FIG. 31 are positive cell populations for both Cy5 and Alexa 488.
  • AuNP-His-Apt-His-BIM was treated in HeLa cells (1 ⁇ 10 7 ) and incubated for 15 or 30 minutes, respectively, trypsizined, centrifuged, and then with phosphate buffered saline (PBS). Washed. Once the trypsin is removed, the cell pellet is fixed in Karnovsky's glutaraldehyde-paraformaldehyde mixture in 0.2 M cacodylate buffer (pH 7.4) at room temperature for about 3 hours, and the pellet is cacodylate buffer (to remove the fixative). pH 7.4).
  • Aptamers were quantitatively analyzed to assess the loading capacity of AuNPs for His-apt or GST-apt.
  • the protein binding ability of AuNP-His-Apt or AuNP-GST-Apt was measured. More specifically, known amounts of His-BIM, His-BCL-xL, His-AcrA, or GST-FOXL2 were developed on SDS-PAGE to be used as a standard. AuNP-Apt (1 nM) in 10 ⁇ l binding buffer was reacted with His-BIM, His-BCL-xL, His-AcrA, or GST-FOXL2 at the indicated concentrations and analyzed by SDS-PAGE. The amount of protein bound to AuNP was measured by the intensity of the band compared to the standard, and the results are shown in FIG. 34. On the other hand, dissociation constants (K D ) were obtained based on the slope calculated from the graph of the right panel.
  • K D dissociation constants
  • HeLa cells were treated with different AuNP-His-Apt concentrations (0, 0.5, 1.0 and 2 nM) and cultured for 12 hours, and then cell viability was measured. The results are shown in FIG. 35. On the other hand, the results (mean ⁇ SEM) were obtained from the results of experiments performed three times.
  • Annexin V-positive killer cells were identified by flow cytometry, and the results are shown in FIG. 35.
  • DAPI was used for nuclear staining, cells were imaged with ax 40 water immersion objective (Scale bar; 20 ⁇ m) and detection of Alexa 488-labeled mouse IgG or Alexa 546-labeled-rabbit IgG was performed. Each protein was identified through.
  • AuNPs bound with both His-tag and GST-tag aptamers confirmed that each of the two proteins (His-BCL-xL and GST-FOXL2) were delivered into the appropriate intracellular compartments.
  • His-BCL-xL and GST-FOXL2 were delivered into the appropriate intracellular compartments.
  • HeLa cells are injected into nude mice to develop cervical cancer, and 0.5 mg / kg AuNP-His-Apt-His-BIM complex is injected into xenograft tumors every two days, over time.
  • Tumor volume (cm 3 ) was calculated using the following equation, and the results are shown in FIG. 37. Meanwhile, the control mice were injected with AuNP-GST-Apt-His-BIM complex
  • AuNP-His-Apt conjugate To confirm His-BIM delivery efficiency to tumor tissue by AuNP-His-Apt conjugate, AuNP-His-Apt loaded with His-BIM protein (Alexa 488-His-BIM) labeled with Alexa 488 was injected. Tumor sections prepared from mice were confirmed by confocal fluorescence microscopy, and the results are shown in FIG. 38. At this time, the scale bar was 200 ⁇ m.
  • TUNEL analysis confirmed increased BIM protein-mediated apoptosis (apoptotic cell death), and the results are shown in FIG. 40. More specifically, dead cells were observed using a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay kit (Roche, USA).
  • Xenograft tumors were generated by intraperitoneally injecting epidermal growth factor receptor (EGFR) -expressing A431 human epidermoid carcinoma cells into nude mice. After 10 days, AuNP- (His-GST) -Apt loaded with only His-BIM, or AuNP- (His-GST) -Apt loaded with both His-BIM and GST-EGF (0.5 mg / kg of body weight) was injected through the tail vein into mice with tumors. At this time, His-BIM and GST-EGF were labeled with Alexa 488 and Texas Red, respectively.
  • EGFR epidermal growth factor receptor
  • mice were sacrificed and organs (brain, liver, spleen, ovary) were recovered, and cross-sections of xenograft tumor tissues were observed by confocal fluorescence microscopy, and the results are shown in FIG. 41.
  • the scale bar was 200 ⁇ m.
  • a strong green signal reflecting efficient BIM protein delivery was observed in the case of AuNP-Apt with both Texas Red labeled EGF and Alexa 488 labeled BIM bound, whereas in the absence of EGF protein Less BIM protein delivery could be confirmed.
  • the gold nanoparticle-aptamer conjugate of the present application can target delivery of protein in vivo .
  • the protein carrier according to the present invention is capable of effectively delivering proteins into cells by specifically binding to proteins to be delivered by aptamers or protein-specific aptamers of various tags bound to gold nanoparticles, and having very low cytotoxicity. Not only harmless to the human body by using the particles, but also reflects light at various wavelengths can easily determine the location in the cell, it is expected to be useful in the diagnosis or treatment of diseases.

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Abstract

La présente invention concerne un domaine de transduction protéique comprenant des nanoparticules d'or et des aptamères se liant aux surfaces des nanoparticules d'or. Le domaine de transduction protéique selon la présente invention devrait être utile pour le diagnostic ou le traitement de maladies puisque le domaine de transduction protéique se lie spécifiquement aux protéines, qui doivent être administrées par le biais de divers aptamères marqués ou aptamères spécifiques de protéines se liant aux nanoparticules d'or, ce qui permet d'administrer efficacement les protéines dans les cellules. Il peut être inoffensif pour les humains du fait de l'utilisation de nanoparticules d'or ayant une très faible cytotoxicité et il permet une confirmation simple de leur position intercellulaire en reflétant des lumières de diverses longueurs d'onde.
PCT/KR2013/011851 2013-02-13 2013-12-19 Domaine de transduction protéique fondé sur un conjugué d'aptamères-nanoparticules d'or et procédé de production correspondant WO2014126332A1 (fr)

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CN114034748A (zh) * 2021-11-08 2022-02-11 商丘师范学院 一种检测胰岛素的电化学适配体传感器及其制备和使用方法

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KR102205032B1 (ko) 2018-10-25 2021-01-19 (주)에스비바이오사이언스 면역 검출용 접합체 및 이를 사용한 면역 검출 방법
KR102221168B1 (ko) 2021-01-05 2021-02-26 주식회사 에스비바이오사이언스 인플루엔자 a 바이러스 검출용 접합체 및 이를 사용한 인플루엔자 a 바이러스 검출 방법
KR102221169B1 (ko) 2021-01-05 2021-02-26 주식회사 에스비바이오사이언스 단일 가닥 dna 결합 단백질과 신호발생물질이 결합된 접합체를 포함하는 면역 검출 키트 및 이를 사용한 면역 검출 방법
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CN114034748A (zh) * 2021-11-08 2022-02-11 商丘师范学院 一种检测胰岛素的电化学适配体传感器及其制备和使用方法

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